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Site-directed mutagenesis of the branchpoint sequence of intron 4 of the human lecithin : cholesterol acyltransferase gene

University of British Columbia

The lecithin:cholesterol acyltransferase (LCAT) gene encodes a plasma enzyme that plays a key role in the metabolism of high-density lipoproteins (HDL). Previous mutations associated with LCAT deficiency syndromes have been identified in the coding regions of the LCAT gene. Recently, our laboratory has found an intron mutation in three patients with a form of LCAT deficiency previously described as fish-eye disease (FED). The in vitro expression of the intron mutant has been shown to result in the intron retention. Since the natural mutation occurs in a putative branchpoint consensus sequence, we hypothesized that the point mutation might disrupt the splicing of the LCAT pre-mRNA. To test the hypothesis, two other novel mutations, i.e., LCAT IVS4-MUT-1 (T->G) and MUT-2 (T->A), were introduced into the same site of the natural mutation (IVS4:T->C22),). After stable transfection of the mutated LCAT minigenes into BHK cells, neither LCAT activity nor LCAT protein could be detected in the culture medium of the IVS4-MUT-1 and MUT-2 cell lines, as was previously described for the natural mutation. To determine the effects of the introduced mutations on the splicing of pre- mRNA, total RNA from transfected BHK cells was used for RT-PCR analysis. All BHK cell lines were shown to transcribe the integrated LCAT minigenes. However, the sizes of these LCAT messages indicated that intron 4 was retained in the IVS4-MUT-1 and MUT-2 cell lines. Subsequent sequence analysis of the RT-PCR products demonstrated that the unspliced intronic sequences contained the introduced mutations, suggesting that the observed retention of intron 4 of the LCAT gene is the result of the specific loss of a thymine residue two bases upstream of the branchpoint adenosine. In attempts to investigate the possible mechanisms responsible for the defective splicing and to study further the functional significance of the branchpoint sequence, a series of mutations was generated in the whole region of the branchpoint sequence. After these intron mutants were transiently expressed in HEK-293 cells, the efficiency of pre-mRNA splicing was analyzed using RT-PCR as well as the measurement of LCAT activity. The results revealed that (1) the mutation of the branchpoint adenosine to any other nucleotide completely abolished the splicing; (2) the insertion of a normal branch site into the intronic sequence of the natural (IVS4-22c) or the branchpoint (IVS4-20t) mutant restored normal splicing; (3) the natural mutation could be partially suppressed by changing its consensus sequence from CCCCGAC to CCCCAAC; and (4) other single-base changes, especially around the branchpoint adenosine residue, significantly decreased the efficiency of splicing and thus enzyme activity. Surprisingly, the nucleotide transversion at the last position of the branchpoint sequence (i.e. IVS4-25a or 25g) resulted in 2.7-fold increase in splicing efficiency. These results have demonstrated that the branchpoint sequence, although only weakly conserved in mammals, can be of essential importance for accurate and efficient splicing of human nuclear pre-mRNA and have contributed to better understanding of the mechanism of branch-site selection during pre-mRNA splicing. The findings also suggest that a DNA polymorphism involving the branchpoint sequence of an intron might affect the efficiency of RNA splicing and thus have significant clinical implications.

Thesis/Dissertation

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2009-07-24

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http://hdl.handle.net/2429/11238

Pathology

University of British Columbia

UBCV

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